The analysis of the elements involved in the control of initiation of viral transcription will be analyzed by a combination of biochemical and genetic approaches. We will purify nascent RNAs by the mercurated nucleotide procedure or from transcriptional complexes. The 5' ends, interspliced regions and regions of the genome from which they originate will be characterized, both at early and late times after Ad 2 infection of HeLa cells. A qualitative and quantitative analysis of initiation and its fidelity in nuclei isolated from Ad 2 infected cells will be performed. These studies will also be extended to mutants (generated by in vitro manipulation of the viral DNA) which are located at or close to the sites of initiation of transcription. These mutants will also be produced in the VA region, in an attempt to define the function of these low molecular weight viral RNAs.